6.9: The Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) can amplify a region of DNA from any source, even from a single cell’s worth of DNA or from fragments of DNA obtained from a fossil. This amplification usually takes just a few hours, generating millions of copies of the desired target DNA sequence. The effect is to purify the DNA from surrounding sequences in a single reaction! Kary B. Mullis was awarded a Nobel Prize in 1993 for his development of PCR, which is now the basis of innumerable research studies of gene structure, function and evolution as well as applications in criminal forensics, medical diagnostics and other commercial uses. PCR is described in detail below.

A. PCR - the Basic Process

Typical PCR relies on knowing two bits of DNA sequence that will be used to design and synthesize short oligonucleotide sequences (oligomers) in the laboratory. The two oligomers are chosen to be complementary to sequences opposite strands of double-stranded DNA containing the gene to be studied. We say that the two oligomers face, or oppose each other. That just means that the 3’ end of one oligomer faces the 3’ end of the opposing oligomer. This way the two oligomers can serve as primers for the elongation replication of both strands of a double stranded target DNA sequence. Check out the link below for further explanation.

272 PCR: Design and Synthesize Opposing Oligonucleotide Primers

The first step in PCR is to add oligomer primers to the target DNA from which a gene (or other genomic sequence) is to be amplified. The mixture is then heated to denature the target DNA. The mixture is cooled to allow the primers to H-bond to complementary target DNA strands. Next, the four deoxynucleotide precursors to DNA (dATP, dCTP, dTTP and dGTP) are added along with a small amount of a DNA polymerase. New DNA strands will now lengthen from the oligonucleotide primers on the template DNAs. To make lots of the PCR product, this reaction cycle must be repeated many times. Therefore, after allowing elongation, the mixture is heated to denature (separate) all the DNA strands. When the mixture is again cooled, the oligomers again find complementary sequences with which to H-bond. Early versions of PCR originally relied on an E. coli DNA polymerase, which is inactivated by heating, and so had to be re-added to the PCR mixture for each elongation cycle. Just as with DNA sequencing, researchers very quickly switched to the heat-stable Taq polymerase, of Thermus aquaticus. The enzymes of T. aquaticus remain active at the very high temperatures at which these organisms live. Since heating does not destroy the Taq polymerase in vitro, PCR, like DNA sequencing reactions, could be automated with programmable thermocylers that raised and lowered temperature required by the PCR reactions. There was no longer a need to replenish a DNA polymerase once the reaction cycles were begun. Thermocyling in a typical PCR amplification is illustrated below for the first two PCR cycles, the second of which, produces the first strands of DNA that will actually be amplified exponentially.

You can see from the illustration that the second cycle of PCR has generated the two DNA strands that will be templates for doubling and re-doubling the desired product after each subsequent cycle. A typical PCR reaction might involve 30 PCR cycles, resulting in a nearly exponential amplification of the desired sequence.

273 PCR: The Amplification Process

Challenge:

Starting with a pair of complementary target DNA molecules (after the 3rd PCR cycle), how many double stranded PCR products should you theoretically have at the end of all 30 PCR cycles?

The amplified products of PCR amplification products are in such abundance that they can easily be seen under fluorescent illumination on an ethidium bromide-stained agarose gel (below).

In this gel, the first lane (on the left) contains a DNA ladder, a mixture of DNAs of known lengths that can be used to estimate the size of the PCR fragments in the 3 rd and 4th lanes (the gel lane next to the ladder is empty). The two bright bands in lanes 3 and 4 are PCR products generated with two different oligomer primer pairs. PCRamplified DNAs can be sequenced and used in many subsequent studies.

B. The Many Uses of PCR

PCR-amplified products can be labeled with radioactive or fluorescent tags to serve as hybridization probes for

• screening cDNA or genomic libraries and isolation of clones.
• determining migration position on a Southern blot.
• determining migration position on a northern blot (a fanciful name for RNAs that are separated by size on gels and blotted to filter).
• and more!

1. Quantitative PCR

We noted above that PCR has wide applications to research, medicine and other practical applications. A major advance was Quantitative PCR, applied to studies of differential gene expression and gene regulation. In Quantitative PCR, initial cDNAs are amplified to detect not only the presence, but also the relative amounts of specific transcripts being made in cells.

2. Forensics

Another application of PCR is in forensic science, to identify a person or organism by comparing its DNA to some standard, or control DNA. An example of one of these acrylamide gel DNA fingerprints is shown below.

Using this technology, it is now possible to detect genetic relationships between near and distant relatives (as well as to exclude such relationships), determine paternity, demonstrate evolutionary relationships between organisms, and on many occasions, solve recent and even ‘cold-case’ crimes. Click Sir Alec Jeffries to learn about the origins of DNA fingerprinting in real life …and on all those TV CSI programs! Check out here for a brief history of the birth of DNA fingerprinting, and to see how analysis of changes in gene activity that occur after death may even help ID criminals. For a video on DNA fingerprinting, click Alu and DNA fingerprinting. Alu is a highly repeated ~300bp DNA sequence found throughout the human genome. Alu sequences are short interspersed elements, or SINES, a retrotransposon we saw earlier. DNA fingerprinting is possible in part because each of us has a unique number and distribution of Alu SINEs in our genome. To read more about Alu sequences and human diversity, click Alu Sequences and Human Diversity.

Intriguing examples of the use of PCR for identification include establishing the identities of Egyptian mummies, the Russian Tsar deposed and killed during the Russian revolution (along with his family members), and the recently unearthed body of King Richard the 3rd of England. Variant PCR protocols and applications are manifold and often quite inventive! For a list, click Variations on Basic PCR.

274 The Power of PCR: Some Examples

3. Who are your Ancestors?

Tracing your ethnic, racial and regional ancestry is related to DNA fingerprinting, in that it relies on PCR amplification of genes and other DNA regions and comparison of these your sequences to distinguishing DNA markers in large sequence databases. The price of these services have come down, and as a result, their popularity has gone up in recent years. Typically, you provide spit or a salivary (buccal) swab to the service and they amplify and sequence the DNA in your samples. The analysis compares your DNA sequences to database sequences looking for patterns of ethnic and regional markers that you might share with the database(s). Based on these comparisons, you are provided with a (more…, or less) accurate map of your DNA-based ancestry. Folks who are spending around $100.00 (less when on sale!) often ask just how accurate are these analyses, and what do they actually mean. For example, what does it mean if your DNA says you are 5% native American? In fact, different services can sometimes give you different results! You can get some answers and explanations DNA Ancestry Testing.